1. Field of the Invention
This invention relates to thrombolytic agents and methods for making and using thrombolytic agents. Specifically, the invention relates to thrombolytic agents comprising a thrombolytic proteinase and a specific binding peptide that specifically binds at the site of blood clots and thrombi in vivo. The invention particularly provides chemically crosslinked conjugates of a thrombolytic proteinase and a plurality of specific binding peptides. Methods for producing such chemically crosslinked conjugates and methods for using such conjugates for eliminating thrombi in vivo to alleviate pathological conditions caused thereby are also provided by the invention.
2. Description of the Related Art
Thrombosis and thromboembolism, the occurrence of occlusive thrombi in the vasculature of human patients, poses a significant clinical problem and is a significant cause of morbidity and mortality. Arterial thrombi are responsible for myocardial infarction (MI) and ischemia (stroke), while venous thrombi cause deep vein thrombosis (DVT) and pulmonary embolism (PE). The magnitude of the clinical challenge created by thrombi is reflected in morbidity and mortality statistics. One of the leading causes of death in men over the age of 50 is acute MI, and stroke remains a debilitating and unpredictable disease. It has been estimated that in the U.S. approximately 5 million patients experience one or more episodes of DVT per year and that over 500,000 cases of PE occur, resulting in 100,000 deaths (Seabold, Society of Nuclear Medicine Annual Meeting 1990).
Anticoagulant therapy can effectively treat these conditions in many cases, if applied early enough. However, such treatment is associated with risks (e.g. internal bleeding) that preclude unnecessary prophylactic application. More advanced techniques of thrombolytic intervention (such as the administration of recombinant tissue plasminogen activator or streptokinase) can be used in acute cases, but these techniques carry even greater risks, due in large part to systemic side effects. In addition, particularly in arterial thrombi, rethrombosis and reocclusion of blood vessels at the thrombus site is a significant (10-30%) clinical outcome. Even using these advanced methodologies, 25-30% of acute MI patients fail to restore perfusion to the thrombus-occluded coronary artery that are the proximal cause of the heart attack.
Thrombi are comprised of blood cells, largely platelets, enmeshed in cross-linked fibrin protein. Thrombus formation involves the conversion of fibrinogen to fibrin and the physiological conversion of unactivated platelets to an activated state. Little fibrin circulates in the bloodstream (in contrast to its precursor, fibrinogen) and most circulating platelets are unactivated, so that fibrin and activated platelets are specifically located at thrombus sites and comprise specific targets for agents designed to dissolve and eliminate thrombi.
Existing pharmaceutical agents for attacking and dissolving thrombi are directed towards enzymatic digestion of the fibrin matrix. Thus, streptokinase, urokinase and tissue plasminogen activator (tPA) are currently in clinical use for dissolving arterial thrombi, particularly arterial thrombi found in cardiac blood vessels. However, the systemic side effects of these agents (systemic fibrinogenolysis and bleeding) raise significant clinical difficulties. These side effects are the result, at least in part, of the fact that these therapeutic agents comprise proteolytic components of the blood clotting cascade, and thus their thrombolytic capabilities are also responsible for their capacity to disrupt hemostasis systemically, particularly at dosages required to result in therapeutically effective delivery of these drugs to the thrombus site. Thus, the outcome of clinical administration of these agents would be improved if a thrombolytic agent was specifically targeted to thrombus sites in vivo, thereby reducing the incidence of unwanted systemic side effects.
Pharmaceutical agents directed towards achieving targeted delivery of thrombolytic agents to thrombus sites in vivo have been reported in the prior art.
Bajwa et al., 1980, Toxicon 18: 285-290 disclose the existence of fibrinolytic enzyme activity in snake venom.
Gartner et al., 1985, J. Biol. Chem. 260: 11891-11894 disclose RGD analogues for inhibiting platelet aggregation and fibrinogen binding.
Plow et al., 1987, Biochem. Pharmacol. 36: 4036-4040 provide a review of platelet-fibrinogen interactions related to thrombus formation.
Bode et al., 1987, J. Biol. Chem. 262: 10819-10823 disclose an antibody-urokinase conjugate for targeting to fibrin.
Retzios et al., 1988, Thromb. Res. 52: 541-552 disclose biochemical characterization of fibrolase.
Dewerchin et al., 1989, Eur. J. Biochem. 185: 141-149 disclose urokinase-antifibrin.sub.XL monoclonal antibody conjugates.
Coller et al., 1989, Circulat. 80: 1766-1774 disclose monoclonal antibodies to platelet GPIIb/IIIa receptors for abolishing thrombus formation in vivo.
Shebuski et al., 1989, Thrombos. Haemostas. 61: 183-188 disclose the use of RGDS peptides as antiaggregatory agents in vivo.
Dennis et al., 1989, Proc. Natl. Acad. Sci. USA 87: 2471-2475 disclose the existence of a class of platelet GPIIb/IIIa antagonists in snake venom.
Shebuski et al., 1990, Circulat. 82: 169-177 disclose the combination of heparin, bitistatin and tissue plasminogen activator as an improved embodiment of a thrombolytic regimen.
Ahmed et al., 1990, Haemostasis 20: 334-340 disclose the thrombolytic properties of fibrolase isoform A.
Charpie et al., 1990, Biochem. 29: 6374-6378 disclose bispecific monoclonal antibody-derived hybrids directed towards fibrin and urokinase.
Runge et al., 1990, Bioconj. Chem. 1: 274-277 disclose bispecific monoclonal antibody-derived hybrids directed towards fibrin and plasminogen activator.
Collen, 1990, Ann. Intern. Med. 112: 529-538 provide a comparison of the benefits of treating myocardial infarction with streptokinase and tissue plasminogen activator.
Rapaport, 1991, Amer. J. Cardiol. 68: 17E-22E provides a review of the use of thrombolytic agents for treatment of thrombi in vascular disease.
Yasuda et al., 1991, Circulat. 83: 1038-1047 disclose a polypeptide platelet GPIIb/IIIa antagonist.
Holahan et al., 1991, Pharmacol. 42: 340-348 disclose the use of echistatin to prevent reocclusion following tissue plasminogen activator thrombolysis.
Dewerchin et al., 1991, Blood 78: 1005-1018 disclose plasminogen activator-antibody conjugates.
Branscomb et al., 1991, Thromb. Haemostas. 64: 260-266 disclose bispecific monoclonal antibody-derived hybrids directed towards fibrin and urokinase.
Runge et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10337-10341 disclose conjugates between an anti-fibrin monoclonal antibody and tissue plasminogen activator.
Kurokawa et al., 1991, Thromb. Haemostas. 66: 684-693 disclose bispecific monoclonal antibody-derived hybrids directed towards fibrin and urokinase.
Guan et al., 1991, Arch. Biochem. Biophys. 289: 197-207 disclose purification and characterization of fibrolase from snake venom.
D'Souza et al., 1991, Trends in Biochem. Sci. 16: 246-250 provide a review of RGD peptides and their role in cell adhesion and thrombus formation.
Abel, 1992, Acta Cardiol. 47: 287-295 provide a review of thrombolysis as a general approach to treating vascular occlusion.
Haber et al., 1992,. Ann. N.Y. Acad Sci. 667: 365-381 disclose the use of antibody targeting as a thrombolytic strategy.
Randolph et al., 1992, Protein Science 1: 590-600 disclose the amino acid sequence of fibrolase.
Neblock, 1992, Bioconj. Chem. 3: 126-131 disclose conjugates between a platelet-specific antibody and tissue plasminogen activator.
Baker et al., 1992, J. Med. Chem. 35: 2040-2048 disclose cyclic RGD peptides as antithrombotic agent.
Du et al., 1993, Sci. China B 36: 1483-1489 disclose a urokinase conjugate to an antibody specific for activated human platelets.
More et al., 1993, Cardiovasc. Res. 27: 2200-2204 disclose conjugates between urokinase and monoclonal antibodies specific for platelet GPIIb/IIIa receptor and damaged endothelium.
Holvoet et al., 1993, Blood 81: 696-703 disclose a conjugate between a fibrin-specific single chain Fv antibody fragment and single-chain urokinase.
Markland et al., 1994, Circulat. 90: 2448-2456 disclose the thrombolytic effects of recombinant fibrolase.
Trika et al., 1994, Thromb. Res. 73: 39-52 disclose purification of platelet GPIIb/IIIa antagonists from snake venom.
Trika et al., 1994, Toxicon 32: 1521-1531 disclose purification of different isoforms of fibrolase to snake venom.
Phaneuf et al., 1994, Thromb. Haemostas. 71: 481-487 disclose streptokinase-hirudin conjugates for thrombolytic targeting.
Bode et al., 1994, Ann. Hematol. 69: S35-S40 provide a general review of thrombolytic approaches to treating myocardial infarction.
In addition to their thrombolytic activity, the thrombolytic agents in current clinical use can also activate thrombin and plasmin at the thrombus site, increasing the likelihood of the re-formation of the thrombus and reocclusion of the vessel. Interaction between platelets and fibrinogen constitutes a critical step in reocclusion, comprising the seeding point for the re-formation of the thrombus. Disruption of the interaction of platelets and fibrinogen would therefore comprise an effective inhibitor to thrombus re-formation.
The interaction between platelets and fibrinogen has been recognized to reside in the platelet glycoprotein IIb/IIIa integrin receptor, which recognizes the amino acid motif -Arg-Gly-Asp found in the fibrinogen protein. This motif therefore comprises a means for specific, targeted disruption of the platelet-fibrinogen interaction.
Peptides and other compounds capable of binding to platelets via the GPIIb/IIIa receptor are known in the prior art.
Ruoslahti & Pierschbacher, U.S. Pat. No. 4,578,079 describe peptides of sequence X-Arg-Gly-Asp-R-Y, wherein X and Y are either H or an amino acid, and R is Thr or Cys, the peptides capable of binding to thrombi in vivo.
Ruoslahti & Pierschbacher, U.S. Pat. No. 4,792,525 describe peptides of sequence Arg-Gly-Asp-X, wherein X is Ser, Thr or Cys, the peptides capable of binding to thrombi in vivo.
Klein et al., 1992, U.S. Pat. No. 5,086,069 disclose guanine derivatives that bind to the GPIIb/IIIa receptor as found on the cell surface of activated platelets.
Pierschbacher et al., 1989, PCT/US88/04403 disclose conformationally-restricted RGD-containing peptides for inhibiting cell attachment to a substratum.
Hawiger et al., 1989, PCT/US89/01742 relates to peptides comprising sequences for two binding sites of a protein.
Nutt et al., 1990, European Patent Application 90202015.5 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application 90202030.4 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application 90202031.2 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application 90202032.0 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application 90311148.2 disclose cyclic peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application 90311151.6 disclose cyclic peptides that are fibrinogen receptor antagonists.
Ali et al., 1990, European Patent Application 90311537.6 disclose cyclic peptides that are fibrinogen receptor antagonists.
Barker et al., 1991, PCT/US90/03788 disclose cyclic peptides for inhibiting platelet aggregation.
Pierschbacher et al., 1991, PCT/US91/02356 disclose cyclic peptides that are fibrinogen receptor antagonists.
Egbertson et al., 1992, European Patent Application 0478328A1 disclose tyrosine derivatives that bind with high affinity to the GPIIb/IIIa receptor.
Ojima et al., 1992, 204th Meeting, Amer. Chem. Soc. Abst. 44 disclose synthetic multimeric RGDF peptides useful in inhibiting platelet aggregation.
Hartman et al., 1992, J Med. Chem. 35: 4640-4642 describe tyrosine derivatives that have a high affinity for the GPIIb/IIIa receptor.
International Patent Application Nos. PCT/US93/04794, PCT/US94/03878 and PCT/US95/06909 to Diatide disclose GPIIb/IIIa binding specific binding peptides.
Thus, there remains a need in the art to target thrombolytic agents specifically to thrombus sites in vivo and thereby reduce or eliminate deleterious systemic side effects resulting from the administration of these agents. There also remains a need to inhibit re-formation of thrombi and re-occlusion of blood vessels with such thrombi after acute resolution of a thrombotic episode, especially in instances of MI. There remains a need for the development of agents capable of fulfilling both roles as specifically-targeted thrombolytic agents and platelet aggregation/fibrinogen interaction antagonists. Chemically crosslinked conjugates of thrombolytic agents and specific binding peptides, and in particular platelet binding peptides, fulfill this need and are provided by this invention as disclosed herein.